Isolation and partial chemical characterization of macrophage-derived neutrophil chemotactic factor

Macrophages stimulated with lipopolysaccharide (LPS) release a factor (MNCF; macrophage-derived neutrophil chemotactic factor) which induces neutrophil migration in vivo and in vitro. The in vivo chemotactic activity of crude MNCF is not affected by pretreating the animals with dexamethasone, an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. We purified MNCF by affinity chromatography of the supernatant from LPS-stimulated macrophages on immobilized D-galactose, followed by gel filtration of the sugar-binding material on Superdex 75. The activity was eluted in the volume corresponding to a MW of 54 kDa. SDS–PAGE of this preparation revealed a single band, also corresponding to a 54 kDa protein. MNCF is an acidic protein (pI < 4) as shown by chromatofocussing. Like the crude MNCF, the homogeneous protein induced neutrophil migration in vitro as well as in vivo. This was not modified by dexamethasone pretreatment

Biological characterization of purified macrophage-derived neutrophil chemotactic factor

We have recently described the purification of a 54 kDa acidic protein, identified as macrophage-derived neutrophil chemotactic factor (MNCF). This protein causes in vitro chemotaxis as well as in vivo neutrophil migration even in animals treated with dexamethasone. This in vivo chemotactic activity of MNCF in animals pretreated with dexamethasone is an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. MNCF is released in the supernatant by macrophage monolayers stimulated with lipopolysaccharide (LPS). In the present study, we describe some biological characteristics of homogenous purified MNCF. When assayed in vitro, MNCF gave a bell-shaped dose–response curve. This in vitro activity was shown to be caused by haptotaxis. Unlike N-formyl-methionylleucyl- phenylalanine (FMLP) or interleukin 8 (IL-8), the chemotactic activity of MNCF in vivo and in vitro, was inhibited by preincubation with D-galactose but not with D-mannose. In contrast with IL-8, MNCF did not bind to heparin and antiserum against IL-8 was ineffective in inhibiting its chemotactic activity. These data indicate that MNCF induces neutrophil migration through a carbohydrate recognition property, but by a mechanism different from that of the known chemokines. It is suggested that MNCF may be an important mediator in the recruitment of neutrophils via the formation of a substrate bound chemotactic gradient (haptotaxis) in the inflamed tissues

Macrophage-derived neutrophil chemotactic factor is involved in the neutrophil recruitment inhibitory activity present in the supernatants of LPS-stimulated macrophages

In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-α and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal−) and bound (D-gal+) fractions, with MNCF being found in the D-gal+ fraction. Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal− fraction. Furthermore, the incubation of the D-gal− fraction with anti-TNF-α plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal+ activity. Overall, these results suggest that the D-gal− inhibitory effect is partially mediated by TNF-α and IL-8, and that MNCF accounts for the inhibition of neutrophil migration in vivo by the D-gal+ fraction

Characterization of the mechanisms underlying the crosstalk between galectins and notch in gastric cancer

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related deaths worldwide. Galectins form a family of β-galactosides binding proteins that recognize a variety of glycan-containing proteins at the cell surface and are overexpressed in various tumors, including gastric cancer. Galectins overexpression as well as changes in their subcellular distribution has been associated with gastric cancer progression and poor prognosis. It is not well understood, however, how the interaction between galectins and glycosylated receptors modulates tumor development and growth. Since Notch receptors and ligands contain glycan structures known to bind galectins, we aim to demonstrate that galectins expression in the tumor microenvironment may interfere with Notch signaling activation during tumor development and progression.\ud \ud Materials and methods\ud Immunoprecipitation procedures with gastric cancer cell line AGS (ATCC CRL-1739) and MKN45 (ACC 409) were used to test for association between galectin-1/-3 and Notch-1 receptor. Furthermore, we transfected AGS cell line with siRNA against galectin-1/-3 or scramble using standard protocols (IDT DNA technologies), stimulate them with immobilized human recombinant delta-4 or Jagged-1 and assessed Notch-1 receptor activation.\ud \ud Results\ud Galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling pathway upon activation by Delta/Jagged ligands. Galectin-1 knockdown alters Notch-1 activation induced by Delta-4 whereas galectin-3 knockdown alters jagged-1-mediated Notch-1 activation. Furthermore, we found that exogenously added galectin-3 can enhance Notch-1 activation by Jagged-1.\ud \ud Conclusion\ud Our results suggest that galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling activation induced by Jagged-1 and Delta-4

Interreality for the management and training of psychological stress: study protocol for a randomized controlled trial

Background. Psychological stress occurs when an individual perceives that environmental demands tax or exceed his or her adaptive capacity. Its association with severe health and emotional diseases, points out the necessity to find new efficient strategies to treat it. Moreover, psychological stress is a very personal problem and requires training focused on the specific needs of individuals. To overcome the above limitations, the INTERSTRESS project suggests the adoption of a new paradigm for e-health - Interreality - that integrates contextualized assessment and treatment within a hybrid environment, bridging the physical and the virtual worlds. According to this premise, the aim of this study is to investigate the advantages of using advanced technologies, in combination with cognitive behavioral therapy (CBT), based on a protocol for reducing psychological stress. Methods/Design. The study is designed as a randomized controlled trial. It includes three groups of approximately 50 subjects each who suffer from psychological stress: (1) the experimental group, (2) the control group, (3) the waiting list group. Participants included in the experimental group will receive a treatment based on cognitive behavioral techniques combined with virtual reality, biofeedback and mobile phone, while the control group will receive traditional stress management CBT-based training, without the use of new technologies. The wait-list group will be reassessed and compared with the two other groups five weeks after the initial evaluation. After the reassessment, the wait-list patients will randomly receive one of the two other treatments. Psychometric and physiological outcomes will serve as quantitative dependent variables, while subjective reports of participants will be used as the qualitative dependent variable. Discussion. What we would like to show with the present trial is that bridging virtual experiences, used to learn coping skills and emotional regulation, with real experiences using advanced technologies (virtual reality, advanced sensors and smartphones) is a feasible way to address actual limitations of existing protocols for psychological stress

Galectin-1 as a potential cancer target

Galectins are a family of structurally related carbohydrate-binding proteins, which are defined by their affinity for poly-N-acetyllactosamine-enriched glycoconjugates and sequence similarities in the carbohydrate recognition domain. Galectin-1, a member of this family, contributes to different events associated with cancer biology, including tumour transformation, cell cycle regulation, apoptosis, cell adhesion, migration and inflammation. In addition, recent evidence indicates that galectin-1 contributes to tumour evasion of immune responses. Given the increased interest of tumour biologists and clinical oncologists in this field and the potential use of galectins as novel targets for anticancer drugs, we summarise here recent advances about the role of galectin-1 in different events of tumour growth and metastasis

Administration of single-dose GnRH agonist in the luteal phase in ICSI cycles: a meta-analysis

<p>Abstract</p> <p>Background</p> <p>The effects of gonadotrophin-releasing hormone agonist (GnRH-a) administered in the luteal phase remains controversial. This meta-analysis aimed to evaluate the effect of the administration of a single-dose of GnRH-a in the luteal phase on ICSI clinical outcomes.</p> <p>Methods</p> <p>The research strategy included the online search of databases. Only randomized studies were included. The outcomes analyzed were implantation rate, clinical pregnancy rate (CPR) per transfer and ongoing pregnancy rate. The fixed effects model was used for odds ratio. In all trials, a single dose of GnRH-a was administered at day 5/6 after ICSI procedures.</p> <p>Results</p> <p>All cycles presented statistically significantly higher rates of implantation (P < 0.0001), CPR per transfer (P = 0.006) and ongoing pregnancy (P = 0.02) in the group that received luteal-phase GnRH-a administration than in the control group (without luteal-phase-GnRH-a administration). When meta-analysis was carried out only in trials that had used long GnRH-a ovarian stimulation protocol, CPR per transfer (P = 0.06) and ongoing pregnancy (P = 0.23) rates were not significantly different between the groups, but implantation rate was significant higher (P = 0.02) in the group that received luteal-phase-GnRH-a administration. On the other hand, the results from trials that had used GnRH antagonist multi-dose ovarian stimulation protocol showed statistically significantly higher implantation (P = 0.0002), CPR per transfer (P = 0.04) and ongoing pregnancy rate (P = 0.04) in the luteal-phase-GnRH-a administration group. The majority of the results presented heterogeneity.</p> <p>Conclusions</p> <p>These findings demonstrate that the luteal-phase single-dose GnRH-a administration can increase implantation rate in all cycles and CPR per transfer and ongoing pregnancy rate in cycles with GnRH antagonist ovarian stimulation protocol. Nevertheless, by considering the heterogeneity between the trials, it seems premature to recommend the use of GnRH-a in the luteal phase. Additional randomized controlled trials are necessary before evidence-based recommendations can be provided.</p

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

<p>Abstract</p> <p>Background</p> <p>The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).</p> <p>Methods</p> <p>A total of 16592 prepared spermatozoa were selected and classified into two groups: Group I, spermatozoa which presented their head attached to an HA substance (HA-bound sperm), and Group II, those spermatozoa that did not attach to the HA substance (HA-unbound sperm). HA-bound and HA-unbound spermatozoa were evaluated according to the following sperm forms: 1-Normal morphology: normal nucleus (smooth, symmetric and oval configuration, length: 4.75+/-2.8 μm and width: 3.28+/-0.20 μm, no extrusion or invagination and no vacuoles occupied more than 4% of the nuclear area) as well as acrosome, post-acrosomal lamina, neck, tail, besides not presenting a cytoplasmic droplet or cytoplasm around the head; 2-Abnormalities of nuclear form (a-Large/small; b-Wide/narrow; c-Regional disorder); 3-Abnormalities of nuclear chromatin content (a-Vacuoles: occupy >4% to 50% of the nuclear area and b-Large vacuoles: occupy >50% of the nuclear area) using a high magnification (8400x) microscopy system.</p> <p>Results</p> <p>No significant differences were obtained with respect to sperm morphological forms and the groups HA-bound and HA-unbound. 1-Normal morphology: HA-bound 2.7% and HA-unbound 2.5% (P = 0.56). 2-Abnormalities of nuclear form: a-Large/small: HA-bound 1.6% vs. HA-unbound 1.6% (P = 0.63); b-Wide/narrow: HA-bound 3.1% vs. HA-unbound 2.7% (P = 0.13); c-Regional disorders: HA-bound 4.7% vs. HA-unbound 4.4% (P = 0.34). 3. Abnormalities of nuclear chromatin content: a-Vacuoles >4% to 50%: HA-bound 72.2% vs. HA-unbound 72.5% (P = 0.74); b-Large vacuoles: HA-bound 15.7% vs. HA-unbound 16.3% (P = 0.36).</p> <p>Conclusions</p> <p>The findings suggest that HA binding assay has limited efficacy in selecting motile spermatozoa with normal morphology at high magnification.</p

Genetic variation among species, races, forms and inbred lines of lac insects belonging to the genus Kerria (Homoptera, Tachardiidae)

The lac insects (Homoptera: Tachardiidae), belonging to the genus Kerria, are commercially exploited for the production of lac. Kerria lacca is the most commonly used species in India. RAPD markers were used for assessing genetic variation in forty-eight lines of Kerria, especially among geographic races, infrasubspecific forms, cultivated lines, inbred lines, etc., of K. lacca. In the 48 lines studied, the 26 RAPD primers generated 173 loci, showing 97.7% polymorphism. By using neighbor-joining, the dendrogram generated from the similarity matrix resolved the lines into basically two clusters and outgroups. The major cluster, comprising 32 lines, included mainly cultivated lines of the rangeeni form, geographic races and inbred lines of K. lacca. The second cluster consisted of eight lines of K. lacca, seven of the kusmi form and one of the rangeeni from the southern state of Karnataka. The remaining eight lines formed a series of outgroups, this including a group of three yellow mutant lines of K. lacca and other species of the Kerria studied, among others. Color mutants always showed distinctive banding patterns compared to their wild-type counterparts from the same population. This study also adds support to the current status of kusmi and rangeeni, as infraspecific forms of K. lacca